研究目的
To develop a fluorescent cocktail nanoparticle for simultaneously detecting intracellular mRNA and H2O2 to differentiate tumor, inflammation, and normal cells for improved tumor diagnosis.
研究成果
The fluorescent cocktail nanoparticle successfully differentiated tumor, inflammatory, and normal cells by cooperative detection of H2O2 and mRNA, showing high sensitivity and specificity with potential for early tumor diagnosis.
研究不足
The study is limited to in vitro cell cultures and may not fully represent in vivo conditions. The specificity and efficiency in complex biological environments need further validation. Potential interferences from other cellular components were not extensively tested.
1：Experimental Design and Method Selection:
The study involved designing a pH-responsive amphiphilic polymer conjugated with an H2O2 probe and encapsulating a DNA probe based on FRET for mRNA detection. Nanoparticles were self-assembled and characterized.
2：Sample Selection and Data Sources:
Cell lines used were NIH3T3 (normal), RAW
3：7 (inflammatory), and HCT116 (tumor) from ATCC. Synthetic DNA sequences were prepared. List of Experimental Equipment and Materials:
2 Instruments included NMR spectrometer, GPC, DLS, TEM, CLSM, flow cytometer, pH meter, and fluorescence spectrometer. Materials included polymers, DNA probes, H2O2, and cell culture reagents.
4：Experimental Procedures and Operational Workflow:
Synthesis of polymers and probes, nanoparticle preparation, characterization (size, zeta potential, pH and H2O2 responsiveness), cell culture, cytotoxicity assays, cellular uptake studies, colocalization experiments, and intracellular imaging via flow cytometry and CLSM.
5：Data Analysis Methods:
Statistical analysis using Student's t-test, fluorescence intensity measurements, and kinetic studies.
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CLSM
Leica
Leica
Confocal laser scanning microscopy for intracellular imaging.
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mPEG113-OH
Sigma-Aldrich
Used in the synthesis of the amphiphilic polymer.
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PMDETA
Sigma-Aldrich
Catalyst in polymerization reactions.
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EDC
Sigma-Aldrich
Coupling agent for chemical synthesis.
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CuBr
Sigma-Aldrich
Catalyst in atom transfer radical polymerization.
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DPA-MA
Sigma-Aldrich
Monomer for polymer synthesis.
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GMA
Sigma-Aldrich
Monomer for polymer synthesis.
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DMAP
Sigma-Aldrich
Catalyst in acylation reactions.
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NHS
Tokyo Chemical Industry Co., Ltd.
Coupling agent for esterification.
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DNase I
Enzyme used to test DNA probe degradation.
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Flow Cytometer
BD LSRFortessa
BD
Used for cellular uptake and fluorescence intensity measurements.
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DLS
Dynamic light scattering for nanoparticle size measurement.
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TEM
Transmission electron microscopy for nanoparticle morphology visualization.
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pH Meter
Used for pH measurements in titration experiments.
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Fluorescence Spectrometer
Used to measure fluorescence spectra.
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Ultrafiltration Device
MWCO = 100 kDa
Used for purifying nanoparticles by removing solvents and free compounds.
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